Phosphoenolpyruvate carboxylase protein kinase from developing castor oil seeds: partial purification, characterization, and reversible control by photosynthate supply

摘要: Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) protein kinase (PPCK) was purified ∼1,500-fold from developing castor oil seeds (COS). Gel filtration and immunoblotting with anti-(rice PPCK2)-immune serum indicated that this Ca2+-insensitive PPCK exists as a 31-kDa monomer. COS PPCK-mediated rephosphorylation of the 107-kDa subunit (p107) PEPC1 (K m = 2.2 μM) activated by ∼80% when assayed under suboptimal conditions (pH 7.3, 0.2 mM PEP, 0.125 mM malate). displayed remarkable selectivity for phosphorylating (relative to tobacco, sorghum, or maize PEPCs), exhibited broad pH-activity optima ∼pH 8.5, at pH 7.3 40–65% 1 mM 10 mM Gln Asn, but inhibited 65% L-malate. The possible control disulfide-dithiol interconversion suggested its rapid inactivation subsequent reactivation incubated oxidized glutathione then dithiothreitol. In vitro activity correlated in vivo p107 phosphorylation status, both peaking mid-cotyledon full-cotyledon COS. Notably, were eliminated following pod excision prolonged darkness intact plants. Both effects fully reversed 12 h reillumination darkened These results implicate direct relationship between up-regulation during recommencement photosynthate delivery illuminated leaves non-photosynthetic Overall, support hypothesis PEPC participate partitioning into C-skeletons needed precursors key biosynthetic pathways